p66Shc has recently emerged as a master regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. While a role for p66Shc as an 'aging'gene that amplifies ROS generation in mitochondria and leads to measurable changes in lifespan in animal models is convincing, there is almost no information on p66Shc functions in cardiomyocytes. Preliminary studies in this application implicate p66Shc as a target of an adrenergic receptor pathway involving ROS and PKC that is localized to caveolae and regulates AKT-FOXO3a phosphorylation and anti-oxidant gene expression. We identify an effect of the G1q-dependent hypertrophy pathway to increase p66Shc expression and target p66Shc to mitochondria where it is predicted to increase ROS generation. We identify a role for protein kinase C-4 (PKC4) and PKC5 to influence p66Shc phosphorylation and localization. p66Shc regulation by PKC4 is particularly noteworthy, as PKC4 also participates in mitochondrial events that control ROS accumulation and apoptosis. Studies in this application will consider the role of p66Shc as a G1q-induced gene product that cooperates with PKC to influence the evolution of cardiac failure phenotypes. Specific aim I will identify p66Shc activation mechanisms and p66Shc signaling functions in cardiomyocytes. The goal is to determine whether p66Shc participates in ROS-regulatory signaling mechanisms in caveolae and mitochondria that influence growth and/or apoptosis. Most studies do not resolve the signaling functions of p66Shc versus the smaller p46/p52Shc isoforms (that do not regulate Redox signaling). Studies in Specific Aim II will use p66Shc knockout models, siRNA gene silencing, and adenoviral mediated overexpression strategies to resolve the specific functions of p66Shc (and critical determinants within the p66Shc protein structure) in mechanisms that regulate ROS production and cardiomyocyte growth/apoptosis. Studies in Specific Aim III will focus on the role of PKC4 to cooperate with p66Shc to control cardiomyocyte remodeling. These studies are based upon preliminary data showing that PKC4 is a p66Shc binding partner that influences p66Shc-S36 phosphorylation and p66Shc subcellular targeting. Finally, studies in Specific Aim IV will examine whether p66Shc gene silencing prevents the structural remodeling and functional deterioration that develops in selected models of cardiac hypertrophy/failure. Evidence that p66Shc contributes to the etiology and/or pathogenesis of cardiac dysfunction would provide the basis for future research focusing on p66Shc as an 'aging gene'that influences the evolution of cardiovascular disorders associated with oxidative stress (ischemic, diabetic, atherosclerotic, hypertensive, and hypertrophic heart disease). The long-term goal of such studies would be to develop novel therapeutic strategies that prevent p66Shc expression or interdict p66Shc function that afford cardioprotection. PUBLIC HEALTH RELEVANCE: Studies in this application focus on p66Shc, an adapter protein that acts as a master regulator of oxidative stress responses and life span in animal models. Based upon the growing awareness that oxidative stress contributes to the evolution of ischemic, diabetic, hypertensive, and/or atherosclerotic heart diseases (i.e., oxidative stress is important regardless of etiology) and our recent studies showing that cardiac hypertrophy leads to increased p66Shc expression and enhanced p66Shc function in cardiomyocytes, studies in this application will examine the role of p66Shc-dependent Redox-regulatory mechanisms in cardiomyocyte growth and apoptosis responses. The long-term goal is to expose novel molecular strategies that can be used to reduce p66Shc expression and/or interdict p66Shc actions to prevent or slow the natural history of clinically important cardiovascular disorders.